Andrew M. Tidball - US grants

DESCRIPTION (provided by applicant): Environmental agents are thought to interact with an individual's genetic makeup to influence or cause neurodegenerative disease. Huntington's disease (HD) is a debilitating neurological condition with a dominant monogenic inheritance pattern. Still, environmental factors are thought to strongly affect disease age at onset and progression. We postulate that the environment causes cellular stress that neurons in the HD brain are susceptible to such as oxidative stress, mitochondrial dysfunction, and calcium dysregulation. We hypothesize that mutant Huntingtin impinges upon the cellular stress response to produce this disease-specific vulnerability. We will test this hypothesis by exposing cells to toxicants known to elicit implicated cellular stress pathways and measure toxicological phenotypes. Here we propose using induced pluripotent stem cells differentiated into striatal neural progenitors from patients with Huntington's disease and controls as our model system. These cell lines contain the mutated Huntingtin gene with the complete promoter region as well as patient- specific genetic background in a neural cell type. In Specific Aim 1, we will optimize the technique for generating DLX2-expressing neural progenitors. We will then expose DLX2-expressing cultures from hiPSCs from two juvenile HD subjects and two control subjects to cell stress model toxicants and evaluate for differential viability. In Specific Aim 2, these same cells will be used to assay the effects toxicant exposure on reactive oxygen species production, mitochondrial membrane potential, ATP content, and cytosolic calcium concentration. The data generated in Aim 1 and Aim 2 will be analyzed by multivariate ANCOVA statistical modeling for disease-toxicant and/or patient-toxicant interactions. For toxicants eliciting a statistically significant disease-toxicant interaction, we will then perform protein and mRNA arrays for important cellular response factors. In Aim 3, any disease-toxicant interactions identified in Aim 1 and Aim 2 will be further tested in hiPSC-derived cells from HD patients with a range of different pathological repeat lengths. Results will also be analyzed by multivariate ANCOVA to determine if any of the phenotypes are repeat length dependent. This proposal will identify types of cellular stress and potential toxicants that could alter the progression of Huntington's disease and investigate the manner by which mutant Huntingtin causes this susceptibility. The proposed research will seek to identify patient-specific risk for these toxicants potentially providing individualized environmental health advice for patients (i.e. personalized environmental medicine). This funding will also contribute to my individual development as an independent researcher by allowing me to pursue these research aims and present the resulting data to the broader scientific community. As the principal investigator, I will have increased ownership of this research project and the freedom to take full advantage of my graduate training by reducing potential financial constraints. PUBLIC HEALTH RELEVANCE: This proposal will use human induced pluripotent stem cells (hiPSCs) from Huntington's disease and control subjects to evaluate the effects of toxicants (e.g. metals, mitochondrial complex inhibitors, and calcium dysregualtors) producing types of cell stress implicated in Huntington's disease progression. We will test cell viability, reactive oxygen species, mitochondrial membrane potential, ATP content, and cytosolic calcium concentration in patient-specific neural progenitors to identify differential toxicity and assess te mechanisms behind these differences. This proposal uses basic science to define potential environmental toxicant modification of Huntington's disease.

Project Summary Most known genetic disorders arise from germline mutations that are present in every cell of the body. However, somatic mutations that occur in neural progenitors during brain development are increasingly recognized as a cause of focal brain malformations and epilepsy. Identifying mutations that cause these focal malformations is extremely challenging because (1) brain tissue from patients must be available and (2) on average only about 5% or fewer of cells in resected cortical tissue contain the causative mutation. For this reason, targeted sequencing of genes expected to cause focal cortical dysplasia (FCD) has been the only successful method for gene identification, and the yield is often very low. We hypothesize that an unbiased cell culture-based, high-throughput screening approach will identify many novel FCD genes. To identify these unknown genes, in Aim 1 we will use a CRISPR interference (CRISPRi) library screen to multiplex knockdown of nearly every gene in cultured human induced pluripotent stem cell (iPSC)-derived, developing excitatory cortical neurons. Positive hits will be isolated by fluorescence-activated cell sorting (FACS) for elevated phopho-S6 (and other dysplastic cell markers) and identified by next-gen sequencing. This screen will allow us to determine which genes, when turned off, lead to FCD-like human brain cell characteristics. In Aim 2, we will perform multiplexed CRISPRi, single-cell RNA-sequencing (direct capture Perturb-seq) for all of the candidate genes found in Aim 1, as well as for all known FCD genes, to both confirm the knockdown of the target gene and compare the transcriptomic effects of each FCD gene candidate. By comparing with known FCD genes, we will identify candidate genes with high confidence and construct an FCD transcriptomic ?fingerprint? that can be used as a resource for further understanding disease mechanisms. In Aim 3, we will apply focal CRISPRi knockdown of DEPDC5, a known FCD gene, to cortical organoids as an in vitro human FCD model. By combining these cutting-edge methods - high-throughput CRISPRi genetic screening, multiplexed gene expression fingerprinting, and brain organoid cultures - these studies will advance our understanding of FCD genetic causes and pathophysiology, providing a platform for future therapeutic studies.